This invention relates to a method of inhibiting the proliferation of cells of certain susceptible leukemias with combinations of (a) recombinant DNA derived human gamma interferon (hIFN-.gamma.) sometimes referred to as human immune interferon and (b) recombinant DNA derived human alpha interferon, which in combination display greater antiproliferative effects against human leukemia cells than is expected from their individual activities.
Gamma interferon has a number of characteristics known in the art that differentiate it from alpha and beta interferons. Among these differences are antigenic distinctiveness and higher levels of immunoregulatory and antitumor activity. Human gamma interferon may be produced by T-lymphocytes stimulated by mitogens or antigens to which they are sensitized. It may also be obtained through cloning and expression techniques now well known in the art.
Although the source of the human gamma interferon used in this invention is not critical, it is required that such hIFN-.gamma. be of a high purity material that is not contaminated by cell constituents or cell debris of the interferon-expressing cell. A preferred hIFN-.gamma. used in this invention is produced by recombinant DNA technology and then purified as taught in Japanese Patent Application No. 281376, filed Dec. 27, 1984 and foreign counterparts thereof, e.g. PCT International Publication No. 8604067 published Jul. 17. 1986. The purification process comprises adding one or more salts of zinc or copper and polyethyleneimine in the extraction. More particularly, it comprises suspending the culture cells of a recombinant microorganism in a buffer solution containing one or more salts of zinc or copper, e.g. zinc chloride, zinc sulfate, zinc acetate, zinc acetylacetonate and copper sulfate, in a range of about 0.5-5 mM in the case of zinc salts and 0.01-3 mM in the case of copper salts, disrupting the cells, then adding polyethyleneimine to the centrifuged supernatant, e.g. to a final concentration of 0.5-1.1%, and subsequently purifying by a conventional method, e.g. combining several chromatographic methods and dialysis.
Human gamma interferon can be made, for example, by the procedures disclosed in Gray, et al., Nature, 295, 503-508 (1982) and Epstein, Nature, 295, 453-454 (1982).
Human alpha interferon is a naturally occurring mixture of at least eleven compounds including those designated alpha-1 interferon and alpha-2 interferon. Alpha interferon exhibiting biological properties similar to those of naturally occurring human or leukocyte interferon can be made by recombinant methods.
A number of alpha interferon species or components are known and are usually designated by a numeral after the Greek letter alpha, and all are contemplated for use in this invention. Thus, the species designated "human alpha-1 interferon" is contemplated for use in this invention and "human alpha-2 interferon" which under USAN, is designated Interferon Alfa-2b, is also contemplated for use in this invention. "Human interferon alfa-2b" is used herein when referring to human alpha-2 interferon. Interferon alfa-2b is the preferred species of human interferon alfa-2.
Human interferon alfa-2 can be produced in bacteria using recombinant techniques. In addition, human interferon alfa-2b may be prepared by recombinant-DNA methods disclosed by Nagata et al., Nature, 284, 316-320 (1980), European Patent 32,134 and U.S. Pat. No. 4,289,690. Various interferon alfa-2 species are disclosed in U.S. Pat. No. 4,503,035. The preferred human interferon alfa-2b used in this invention is also denoted "hIFN-.alpha.2b".
To date, much has been learned about the effect of the interferons on cell proliferation.
Borden et al., Progress in Hematology, Vol XII, Brown, E. B., editor, pp 299-339 (1981) found that interferons inhibit proliferation of normal and transformed cells in vitro.
Gresser et al., Biochim. Biophys. Acta, 516, 231-247 (1978) and Chemotherapy-A Comprehensive Treatise, Becker, F. editor, Vol. 5, pp 521-571 (1977) used murine systems and demonstrated that relatively impure interferon in vitro results in a decreased rate of cell proliferation, lower cell-saturation densities and decreased colony formation in agarose. Inhibitory effects were observed using mouse L929, L1210, Ehrlich ascites, primary embryo and primary weanling kidney cells.
Different types of cells differ in sensitivity to interferons, and cell inhibitory concentrations of interferons can vary widely, i.e. from 10-1000 units, see Borden et al. supra. Among the tumor cells of established cell lines studied in vitro, Einhorn et al., Human Interferon Production and Clinical Use, Stinebring et al., editors, pp 159-174 (1977), found lymphoid leukemias to be sensitive to interferons. The interferons used in these experiments were unpurified materials and it is not clear if the impurities affected the test results.
European Patent Application, Publication No. 0107498, Oct. 24, 1983, discloses combinations of purified human gamma interferon and human alpha interferon made by recombinant methods which exhibit activities higher than could have been fairly predicted based on their respective activities when tested alone. The tests were conducted using the human melanoma cell line Hs294T. However, there is no disclosure that leukemias are susceptible to such treatment.